US7375098B2 - 8β-vinyl-11β-(ω-substituted)alkyl-estra-1,3,5(10)-trienes - Google Patents

8β-vinyl-11β-(ω-substituted)alkyl-estra-1,3,5(10)-trienes Download PDF

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US7375098B2
US7375098B2 US10/829,390 US82939004A US7375098B2 US 7375098 B2 US7375098 B2 US 7375098B2 US 82939004 A US82939004 A US 82939004A US 7375098 B2 US7375098 B2 US 7375098B2
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estra
vinyl
triene
diol
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Nico Braeuer
Olaf Peters
Alexander Hillisch
Rolf Bohlmann
Margit Richter
Hans-Peter Muhn
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Bayer Pharma AG
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Schering AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J5/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/16—Masculine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/30—Oestrogens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J75/00—Processes for the preparation of steroids in general

Definitions

  • This invention relates to 8 ⁇ -vinyl-11 ⁇ -( ⁇ -substituted)alkyl-estra-1,3,5(10)-trienes with ER ⁇ -antagonistic activity, process for their production, their intermediate products, pharmaceutical preparations that contain the compounds according to the invention, as well as their use for the production of pharmaceutical agents.
  • the compounds according to the invention are those steroidal tissue-selective estrogens that have in vitro a higher affinity to estrogen receptor preparations from rat prostates than to estrogen receptor preparations from the rat uterus and that exert in vivo a contraceptive action by their preferential action on the ovary and are distinguished by an improved physicochemical profile.
  • Contraceptive methods with chemical compounds are common in women who do not want to become pregnant.
  • the following chemical methods of female contraception are currently available:
  • Oral contraceptives that consist of the most varied combinations of an estrogen with a gestagen are the most frequently used contraceptives of women. They act according to the endocrine principle. Although such contraceptives are very effective, undesirable side effects can occur, such as, e.g.: irregular bleeding, nausea, vomiting, depression, weight gain or headaches. More serious diseases are also sometimes observed such as thromboembolisms, stroke, liver adenomas, gallbladder diseases or high blood pressure. These undesirable side effects of the oral contraceptives now used make clear the medical need for a new contraceptive method without side effects.
  • An ideal contraceptive method is a method that operates directly on the ovarian follicle without influencing the endocrine hypothalamus-pituitary gland-ovary axis. This could be achieved with a chemical compound that impairs the folliculogenesis, for example by destroying a paracrine interaction between the egg cells and the granulosa cells and thus sees to it that
  • Follicular growth is the development of an ovarian follicle from the primordial stage up to the large antral crack-free follicle. Only an optimally built-up antral follicle has the potential to ovulate a mature egg cell.
  • Gonadotropins such as, e.g., the follicle-stimulating hormone (FSH), however, are mainly involved in the late steps of folliculogenesis, i.e., the development of early antral to the large ovulatory follicle. Also, however, in late folliculogenesis, additional modulators of folliculogenesis are discussed (Elvin et al., Mol. Cell. Endocrinol. 1999, 13, 1035-1048).
  • FSH follicle-stimulating hormone
  • ER ⁇ estrogen receptor- ⁇
  • ER ⁇ While an expression of ER ⁇ could be detected in almost all organs studied, the highest expression of ER ⁇ was found in female animals in the ovary and in male animals in the prostate (Couse et al., Endocrinology 1997, 138, 4613-4621). In the ovary, a significant ER ⁇ expression in the follicles is shown in almost all development stages. While in the follicles, ER ⁇ is expressed only in the outer follicle cells (theca cells), a strong expression of ER ⁇ is present in the estradiol-producing granulosa cells.
  • ER ⁇ is functional in the male animal also follows by studies on ER ⁇ -(ERKO) or ER ⁇ -( ⁇ ERKO)-knockout mice: male ERKO mice (R. A. Hess et al., Nature 1997, 390, 509-512) exhibit significant fertility disorders. To this end, the important function of estrogens with respect to maintaining the testis function relative to fertility is confirmed.
  • ER ⁇ and ER ⁇ have significantly different amino acid sequences in their ligand-binding and transactivating domains. This suggests that
  • new steroidal compounds based on the building block of the unsubstituted estradiol in 8-position, which carry a hydrocarbon radical in 11 ⁇ -position, which contains an individual linear chain with a length of 5 to 9 carbon atoms, are described. These compounds have an ER ⁇ -agonistic/ER ⁇ -antagonistic profile of action. Based on this mixed estrogen receptor profile, these compounds are suitable as improved estrogens for the treatment of estrogen-produced disorders and for contraception together with a gestagen.
  • WO 01/77138 discloses 11 ⁇ -n-pentyl-und 11 ⁇ -n-hexyl-8 ⁇ -substituted estra-1,3,5(10)-trienes with ER ⁇ -antagonistic action.
  • the 11 ⁇ -n-alkyl substitution results in further reduction in polarity and thus also in poorer water-solubility of such compounds.
  • the object of this invention is to provide compounds with improved physicochemical properties that have in vitro a dissociation with respect to the binding to estrogen receptor preparations of rat prostates and rat uteri and exert a contraceptive action in vivo by their preferred action on the ovary, without influencing other estrogen-sensitive organs, such as, e.g., the uterus or the liver. These compounds are also to be used for male contraception as well as for treating benign or malignant proliferative diseases of the ovary.
  • This invention relates to compounds of general formula I
  • R 3 represents a group R 19 —O—, R 20 SO 2 —O—, or —O—C(O)R 21 ;
  • n 3, 4, or 5:
  • This invention also comprises the pharmaceutically compatible salts of the compounds of general formula I according to the invention.
  • the unbranched C 1 -C 8 -alkyl group can be, for example, a methyl-, ethyl-, n-propyl-, n-butyl-, n-pentyl-, n-hexyl-, n-heptyl-, or n-octyl-;
  • the branched C 3 -C 8 -alkyl group is an iso-propyl-, iso-butyl-, sec-butyl, tert-butyl-, iso-pentyl-, neo-pentyl-, 2-methylpentyl-, 2,2-dimethylbutyl-, 2,3-dimethylbutyl-, 2-methylhexyl-, 2,2-dimethylpentyl-, 2,2,3-trimethylbutyl-, or 2,3,3-trimethylbutyl group.
  • the C 3 -C 6 -cycloalkyl groups that are optionally substituted with a phenyl radical can readily be a cyclopropyl-, cyclobutyl-, cyclopentyl-, cyclohexyl-, or phenylcyclopropyl-, phenylcyclobutyl-, phenylcyclopentyl-, or phenylcyclohexyl group.
  • the (C 3 -C 6 -cycloalkyl)-C 1 -C 4 -alkylene group can be, for example, a cyclopropylmethyl-, cyclobutylmethyl-, cyclopentylmethyl-, cyclohexylmethyl, cyclopropylethyl-, cyclobutylethyl-, cyclopentylethyl-, cyclohexylethyl-, cyclopropylpropyl-, cyclobutylpropyl-, cyclopentylpropyl-, cyclohexylpropyl-, cyclopropylbutyl-, cyclobutylbutyl, cyclopentylbutyl- or cyclohexylbutyl group.
  • the branched or unbranched C 2 -C 5 -alkenyl group can be, for example, a vinyl-, trifluorovinyl-, allyl-, homoallyl-, (E)-but-2-enyl-, (Z)-but-2-enyl-, (E)-but-1-enyl-, (Z)-but-1-enyl-, pent-4-enyl-, (E)-pent-3-enyl-, (Z)-pent-3-enyl-, (E)-pent-2-enyl-, (Z)-pent-2-enyl-, (E)-pent-1-enyl-, (Z)-pent-1-enyl-, 2-methylvinyl-, 3-methylbut-3-enyl-, 2-methylbut-3-enyl-, (E)-2-methylbut-2-enyl-, (Z)-2-methylbut-2-enyl-, or 3-methylbut-2-enyl-group.
  • the C 2 -C 5 -alkinyl group can be, for example, an ethinyl-, prop-1-inyl-, prop-2-inyl-, but-1-inyl-, but-2-inyl-, but-3-inyl-, pent-1-inyl-, pent-2-inyl-, pent-3-inyl-, pent-4-inyl-, 1-methylprop-2-inyl-, 1-methylbut-3-inyl-, or 1-ethylprop-2-inyl group.
  • the R 19 O group accordingly can be, for example, a methoxy-, ethoxy-, n-propoxy-, iso-propoxy-, n-butoxy-, sec-butoxy-, iso-butoxy-, or tert-butoxy group.
  • An aryl group can be, for example, a phenyl-, naphthalen-1-yl-, naphthalen-2-yl-, [1,1′-biphenyl]-2-yl-, [1,1′-biphenyl]-3-yl group or a [1,1′-biphenyl]-4-yl group.
  • the heteroaryl group can be a pyridinyl-, pyrimidinyl-, quinolinyl-, isoquinolinyl-, benzofuranyl-, benzothienyl-, 1,3-benzodioxolyl-, 2,1,3-benzothiadiazolyl-, indolyl-, furanyl-, thienyl-, oxazolyl-, isoxazolyl-, thiazolyl-, pyrrolyl-, pyrazolyl-, pyrazinyl-, pyridazinyl group or an imidazolyl group that is linked by one of the points that can be substituted.
  • the heterocyclyl group for radicals Z and Z′ can be a piperidinyl-, morpho-linyl-, thiomorpholinyl-, piperazinyl-, tetrahydrofuranyl-, tetrahydrothienyl-, imidazolidinyl group or a pyrrolidinyl group that can be linked by one of the points that can be substituted.
  • the substituent of the aryl, heteroaryl and heterocyclyl radicals can be, i.a., unbranched or branched C 1 -C 4 -alkyl groups (methyl-, ethyl-, n-propyl-, iso-propyl-, n-butyl-, sec-butyl-, iso-butyl- as well as tert-butyl-) and/or C 2 -C 6 -alkenyl groups (vinyl-, allyl-, homoallyl-, (E)-but-2-enyl-, (Z)-but-2-enyl-, pent-4-enyl-, (E)-pent-3-enyl-, (Z)-pent-3-enyl-, (E)-pent-2-enyl-, (Z)-pent-2-enyl-, 2-methylvinyl-, 3-methylbut-3-enyl-, 2-methylbut-3-enyl-, 2-methyl
  • the aryl-C 1 -C 4 -alkylene group for radicals Z and Z′ can be a combination of the above-defined aryl and C 1 -C 4 -alkyl groups, for example: a phenylmethyl-, 1-phenylethyl-, 2-phenylethyl-, 1-methyl-1-phenylethyl-, 3-phenylpropyl-4-phenylbutyl-, (naphthalen-1-yl)methyl-, 1-(Naphthalen-1-yl)ethyl-, 2-(naphthalen-1-yl)-ethyl-, (naphthalen-2-yl)methyl-, 1-(naphthalen-2-yl)ethyl-, 2-(naphthalen-2-yl)ethyl-, ([1,1′-biphenyl]-2-yl)methyl-, ([1,1′-biphenyl]-3-yl)methyl- or a ([1,1′-b
  • the heteroaryl-C 1 -C 4 -alkylene groups for radicals Z and Z′ can be a combination of the above-defined heteroaryl- and C 1 -C 4 -alkylene groups, for example a (pyridin-2-yl)methyl-, (pyridin-3-yl)methyl-, (pyridin-4-yl)methyl-, (furan-2-yl)methyl-, (furan-3-yl)methyl-, (thien-2-yl)methyl-, (thien-3-yl)methyl-, 2-(thien-2-yl)ethyl- or a 2-(thien-3-yl)ethyl group.
  • halogen is fluorine, chlorine, bromine or iodine.
  • inorganic acids i.a., hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, nitric acid
  • carboxylic acids i.a., acetic acid, propionic acid, hexanoic acid, octanoic acid, decanoic acid, oleic acid, stearic acid, maleic acid, fumaric acid, succinic acid, benzoic acid, ascorbic acid, oxalic acid, salicylic acid, tartaric acid, citric acid, lactic acid, glycolic acid, malic acid, mandelic acid, cinnamic acid, glutaminic acid, asparaginic acid; as sulfonic acids, i.a., methanesulfonic acid, ethanesulfonic acid, toluenesul
  • carboxylic acids i.a., acetic acid, propionic acid, hexanoic acid, octanoic acid, decan
  • Preferred according to this invention are those compounds of general formula I, in which
  • the compounds according to the invention are suitable for inhibiting folliculogenesis and ovulation, for male contraception and for treating benign and malignant proliferative diseases of the ovary.
  • the compounds of general formula I according to the invention can be used alone for contraception, i.e., without the additional administration of gestagens.
  • ester derivatives of the estratrienes according to the invention can have advantages as prodrugs compared to the unesterified active ingredients with respect to their method of administration, their type of action, strength of action and duration of action.
  • the sulfamate derivatives of the estratrienes according to the invention also have pharmacokinetic and pharmacodynamic advantages. Such effects were already described in other steroid-sulfamates (J. Steroid Biochem. Molec. Biol. 1995, 55, 395-403; Exp. Opinion Invest. Drugs 1998, 7, 575-589).
  • This invention describes 8 ⁇ -vinyl-11 ⁇ -( ⁇ -substituted)alkyl-estra-1,3,5(10)-trienes, which have in vitro dissociation with respect to binding to estrogen receptor preparations of rat prostates and rat uteri and which have in vivo preferably an inhibition of folliculogenesis and ovulation.
  • the compounds according to the invention have a contraceptive action over a broad dose range, without influencing other estrogen-sensitive organs, such as, e.g., the uterus or the liver.
  • these compounds can be used for male contraception and for treating benign or malignant proliferative diseases of the ovary.
  • This invention therefore relates to pharmaceutical preparations that contain at least one compound of general formula I as well as their physiologically compatible salts, the use of the compounds of general formula I for the production of a pharmaceutical agent for male and/or female contraception, and for the treatment of benign and malignant proliferative diseases of the ovary.
  • the compounds according to the invention can be used for the following indications both after oral and parenteral administration.
  • the compounds of general formula I according to the invention can be used as individual components in pharmaceutical preparations or in combination especially with GnRH antagonists, progesterone receptor antagonists, mesoprogestins, gestagens or tissue-selective gestagens (action on A/B-form type).
  • the compounds according to the invention and the preparations that contain them are especially suitable for ovarian contraception, for the treatment of benign or malignant proliferative diseases of the ovary, such as, e.g., ovarian cancer, and granulosa cell tumors.
  • the compounds can be used for treating male fertility disorders and prostatic diseases.
  • the amount of a compound of general formula I that is to be administered varies within a wide range and can cover any effective amount.
  • the amount of the compound that is administered can be 0.01 ⁇ g/kg-100 mg/kg of body weight, preferably 0.04 ⁇ g/kg-1 mg/kg of body weight, per day.
  • this corresponds to a dose of 0.8 ⁇ g to 8 g, preferably 3.2 ⁇ g to 80 mg, daily.
  • a dosage unit contains 1.6 ⁇ g to 2000 mg of one or more compounds of general formula I.
  • the compounds according to the invention and their acid addition salts are suitable for the production of pharmaceutical compositions and preparations.
  • the pharmaceutical compositions or pharmaceutical agents contain as active ingredients one or more of the compounds according to the invention or their acid addition salts, optionally mixed with other pharmacologically or pharmaceutically active substances.
  • the production of the pharmaceutical agents is carried out in a known way, whereby the known and commonly used pharmaceutical adjuvants as well as other commonly used vehicles and diluents can be used.
  • the compounds according to the invention can be administered orally or parenterally, for example intraperitoneally, intramuscularly, subcutaneously or percutaneously, or else implanted in the tissue.
  • the dosage units can contain a pharmaceutically compatible vehicle, such as, for example, starch, sugar, sorbitol, gelatin, lubricant, silicic acid, talc, etc.
  • a pharmaceutically compatible vehicle such as, for example, starch, sugar, sorbitol, gelatin, lubricant, silicic acid, talc, etc.
  • the active ingredients can be dissolved or suspended in a physiologically compatible diluent.
  • diluents very often oils with or without the addition of a solubilizer, a surfactant, a suspending agent or an emulsifying agent are used. Examples of oils that are used are olive oil, peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil.
  • the compounds can also be used in the form of a depot injection or an implant preparation, which can be formulated so that a delayed release of active ingredient is made possible.
  • implants can contain, for example, biodegradable polymers, or synthetic silicones such as, for example, silicone rubber.
  • active ingredients can be added to, for example, a patch.
  • intravaginal systems e.g., vaginal rings
  • intrauterine systems e.g., pessaries, coils, IUDs
  • various polymers are suitable, such as, for example, silicone polymers, ethylene vinyl acetate, polyethylene or polypropylene.
  • the compounds can also be formulated as cyclodextrin clathrates.
  • the compounds are reacted with ⁇ -, ⁇ -, or ⁇ -cyclodextrin or derivatives of the latter (PCT/EP95/02656).
  • the compounds of general formula I according to the invention can also be encapsulated with liposomes.
  • FIG. 1 depicts a general operating scheme for synthesis of Aldehyde 9a
  • FIG. 2 depicts a general operating scheme for synthesis of Aldehyde 9b
  • FIG. 3 depicts a general operating scheme for synthesis of Aldehyde 9c
  • FIG. 4 depicts a General Operating scheme
  • FIG. 5 depicts a General Operating scheme
  • FIG. 6 depicts a general operating scheme
  • FIG. 7 depicts a general operating scheme for the introduction of an amino Group and for acylation of amines
  • FIG. 8 depicts a general operating scheme for the oxidation of Aldehydes.
  • the binding affinity of the compounds according to the invention was tested in competitive experiments with use of 3H-estradiol as a ligand on estrogen receptor preparations from rat prostates and rat uteri.
  • the preparation of prostate cytosol and the estrogen receptor test with prostate cytosol was carried out as described by J. Testas et al. in Endocrinology 1981, 109, 1287-1289.
  • the compounds according to the invention have higher binding affinity to the estrogen receptor from rat prostates than to estrogen receptors from rat uteri (Tables 1 and 2).
  • Tables 1 and 2 it is assumed that ER ⁇ predominates in the rat prostates over ER ⁇ , and ER ⁇ predominates in rat uteri over ER ⁇ .
  • Table 1 shows that the ratio of the binding to prostate and uterus receptors qualitatively coincides with the quotient of relative binding affinity (RBA) to human ER ⁇ and ER ⁇ of rats (according to Kuiper et al. Endocrinology 1996, 138, 863-870) (Table 1).
  • U-20S cells are cultivated in Dulbecco 's medium (DMEM) without phenol red (Gibco BRL; #11880-028)+5% fetal calf serum (FCS) (Seromed; #S 0115) ⁇ 100 units/ml of penicillin/100 ⁇ g/ml of streptomycin (Seromed; #A 2213), 4 mmol of L-glutamine (Gibco BRL; #25030-024) (PSG) at 37° C. and 8.5% CO 2 .
  • DMEM Dulbecco 's medium
  • FCS fetal calf serum
  • the ER ⁇ -expression plasmid (HEGO) that was used was amplified in E. coli DH5 ⁇ (Invitrogen Company).
  • the ER ⁇ -expression plasmid (ER ⁇ 0) that was used was produced in-house and amplified in E. coli DH5 ⁇ .
  • the expression plasmid pSG5 was used.
  • the vector pBL-LUC + was provided with two Tandem-EREs (estrogen-responsive elements of the vitellogenin promoter) and amplified in E. coli (XL1-Blue; Stratagene Company).
  • Plasmid-DNA is prepared by means of the NucleoBond Plasmid Maxi Kit (CLONTECH; #K3003-2) and FuGENE 6 reagent (Boehringer Mannheim; #1 814 443). The latter are first diluted separately in a suitable volume of DMEM and incubated, before the solutions are combined and again incubated.
  • DNA Mixture in the 50 ml-BlueMax (Falcon; #2070) tube pSG5-ER ⁇ / ⁇ FL (Heg0/ER ⁇ 0) 2 ng/well (Estrogen receptor- expression plasmid) p(ERE) 2 -luc + 100 ng/well (Luciferase-reporter plasmid) Transfection Reagent (B): in a 14 ml-polypropylene tube (Falcon; #2059) DMEM or Serum (introduction) 9.7 ⁇ l/well FuGENE 6 (directly into the 0.3 ⁇ l/well medium)
  • dilution series for reference and test substances are produced in 96-well plates (Costar; #3595), starting from 10 ⁇ 3 M stock solutions dissolved in dimethyl sulfoxide (DMSO, Sigma; #D-2650).
  • DMSO dimethyl sulfoxide
  • the 10 ⁇ 3 M DMSO solutions are stored at ⁇ 20° C. and must be readily dissolved before the removal (15 minutes, 37° C.).
  • the dilution stages are selected such that the final concentrations on the test plate for agonism lie in the range of 10 ⁇ 7 -10 ⁇ 12 M (for E 2 : 10 ⁇ 8 -10 ⁇ 13 M).
  • the transfection medium is replaced by 180 ⁇ l of D-MEM+5% CCS+PSG per well.
  • the cells are treated in addition with estradiol. Then, 20 ⁇ l of the substance dilutions is pipetted through. The negative controls receive 20 ⁇ l of DMEM+1% DMSO per well. The final test substance concentrations are 3 ⁇ 10 ⁇ 11 M for ER ⁇ or 3 ⁇ 10 ⁇ 10 M for ER ⁇ .
  • the known antiestrogen fulvestrant (AstraZeneca) is used at the same concentration (Table 2).
  • the incubation is carried out overnight (16-18 hours) at 8.5% CO 2 and 37° C.
  • the lysates are mixed with 30 ⁇ l of luciferase substrate A (PharMingen; #556867) and 30 ⁇ l of luciferase substrate B (PharMingen; #556869).
  • the measurement of the luciferase activity is carried out 30 seconds after substrate B is added in the cycle mode of the luminometer (DYNATECH; ML3000).
  • the evaluation of the measurement data is carried out by means of software assigned by the equipment manufacturer (BioLinx).
  • a calculation of EC 50 , the efficacy and the EMR value for the agonism or the IC 50 and the efficacy for the antagonism is possible by means of “MTS” software (Table 2).
  • Immature female rats are treated from day 1 to day 4 with a combination of cetrorelix and the ER ⁇ -selective estrogen 8 ⁇ -vinylestra-1,3,5(10)-triene-3,17 ⁇ -diol (25 mg/kg, s.c.).
  • the vehicle or the active substance is administered in various dosages (1; 3; 10; 30 mg/kg, s.c.) from day 1 to day 4.
  • the autopsy of the animals is carried out on day 5.
  • the ovary is removed and analyzed macroscopically, e.g., organ weights, and microscopically, e.g., histological evaluation of the follicles, so-called follicle staging.
  • Immature female rats are hypophysectomized. This day is defined as day 0. From day 1-day 4, subcutaneous and/or oral treatment is carried out with the active substance in combination with 17 ⁇ -estradiol. On day 5, a subcutaneous injection with PMSG (pregnant mare serum gonadotropin) is carried out. On day 7, hCG is administered intraperitoneally to trigger ovulation. On day 8, the ovary is removed and analyzed macroscopically (e.g., ovary weight) and/or microscopically (e.g., histological evaluation of the follicles, so-called follicle staging). The tubes are flushed and checked for the presence of egg cells.
  • PMSG pregnant mare serum gonadotropin
  • immature female rats are treated subcutaneously with PMSG (pregnant mare serum gonadotropin) (day 1).
  • PMSG pregnant mare serum gonadotropin
  • the animals receive the active substance, administered subcutaneously or orally.
  • the animals receive an intraperitoneal injection of hCG to trigger ovulation.
  • Autopsy is carried out 16 hours after the hCG is administered. The tubes are flushed and checked for the presence of eggs cells.
  • Another possibility to detect in vivo the dissociated estrogen action of the substances according to the invention consists in the fact that after a one-time administration of the substances in rats, effects on the expression of 5HT2a-receptor and serotonin transporter protein and mRNA levels in ER ⁇ -rich brain areas can be measured. Compared to the effect on the serotonin receptor and transporter expression, the effect on the LH-secretion is measured. Substances with higher binding to the rat prostate—compared to the rat uterus estrogen receptor—are more potent with respect to increasing the expression of serotonin receptors and transporters, in comparison to their positive effect on the LH release.
  • the density of serotonin receptors and transporters is determined in brain sections using radioactive ligands, and the corresponding mRNA is determined using in situ hybridization. The method is described in the literature: G. Fink & B. E. H. Sumner 1996 Nature 383: 306; B. E. H. Sumner et al. 1999 Molecular Brain Research, in press.
  • This invention also relates to the intermediate products of general formula VI
  • radicals X, R 3 , R 17 , R 17′ and n have the same meaning as in general formula I.
  • this invention also relates to a process for the production of the compounds of general formula I, as well as, in each case, processes for the production of individual intermediate stages VI to IX.
  • the compounds of general formula I according to the invention can be produced as described in the Examples.
  • the additional compounds of general formula I can be obtained.
  • Etherification and/or esterification of free hydroxy groups is carried out according to methods that are common to one skilled in the art.
  • the compounds according to the invention can be present at carbon atom 17 as ⁇ , ⁇ -stereoisomers.
  • the compounds In the production of compounds according to the described processes, the compounds accumulate in most cases as mixtures of the corresponding ⁇ , ⁇ -isomers.
  • the mixtures can be separated by, for example, chromatographic processes.
  • 17-Substituents are also introduced according to known processes by nucleophilic addition of the desired substituent or a reactive precursor thereof and optionally further built up.
  • estratriene-carboxylic acid esters according to the invention are produced from the corresponding hydroxy steroids analogously to processes that are also known (see, e.g., Pharmazeutician Wirkstoffe, Synthesen, Patente, füren [Pharmaceutical Active Ingredients, Syntheses, Patents, Applications]; A. Kleemann, J. Engel', Georg Thieme Verlag Stuttgart 1978. Arzneistoff, Fort Coloure [Pharmaceutical Agents, Improvements] 1972 to 1985; A. Kleemann, E. Lindner, J. Engel, VCH 1987, pp. 773-814).
  • estratriene-sulfamates according to the invention are available in a way that is known in the art from the corresponding hydroxy steroids by esterification with sulfamoyl chlorides in the presence of a base (Z. Chem. 1975, 15, 270-272; Steroids 1996, 61, 710-717).
  • the regioselective esterification of polyhydroxylated steroids with N-substituted and N-unsubstituted sulfamoyl chlorides is carried out according to partial protection of those hydroxyl groups that are to remain unesterified.
  • Silyl ethers have turned out to be protective groups with selective reactivity that is suitable for this purpose, since these silyl ethers are stable under the conditions of sulfamate formation, and the sulfamate group remains intact when the silyl ethers are again cleaved off for regeneration of the residual hydroxyl group(s) still contained in the molecule (Steroids 1996, 61, 710-717).
  • the production of the sulfamates according to the invention with one or more additional hydroxyl groups in the molecule is also possible in that the starting material is suitable hydroxy-steroid ketones.
  • the starting material is suitable hydroxy-steroid ketones.
  • one or more hydroxyl groups that are present are subjected to sulfamoylation.
  • the sulfamate groups optionally can be converted with a desired acyl chloride in the presence of a base into the (N-acyl)sulfamates in question.
  • oxosulfamates or oxo-(N-acyl)sulfamates are converted by reduction into the corresponding hydroxysulfamates or hydroxy-(N-acyl)sulfamates (Steroids 1996, 61, 710-717).
  • Sodium borohydride and the borane-dimethyl sulfide complex are suitable as proper reducing agents.
  • Substituents according to general formula I can also be introduced in the stage of estratrienes that are already substituted in 8-position, however. This can be useful or necessary especially in the case of multiple substitutions of the desired final compound.
  • 11-keto-estratetraene derivatives (U.S. Pat. No. 3,491,089, Tetrahedron Letters, 1967, 37, 3603), which are substituted stereoselectively in 8 ⁇ -position in the reaction with diethylaluminum cyanide, are used.
  • the synthesis of compound (1) is described (WO 01/77139).
  • 8 ⁇ -substituted 11-alkyl-estra-1,3,5(10),9(11)-tetraenes are obtained.
  • the 8 ⁇ -cyano grouping can then be converted into the 8 ⁇ -aldehyde.
  • a functionalization e.g. by Wittig reactions
  • after hydrogenation of the C(9)-C(11) double bond has taken place, results in the 8 ⁇ ,11 ⁇ -disubstituted steroids according to the invention (Diagram 1).
  • the 8 ⁇ -substituted 11-alkyl-estra-1,3,5(10),9(11)-tetraenes that are first obtained in this sequence, just like the 8 ⁇ -substituted 11 ⁇ -alkyl-estra-1,3,5(10),9(11)-trienes, can be further reacted to many substitution patterns on the steroid according to the method that is known to one skilled in the art.
  • Tf Trifluoromethanesulfonyl
  • TBS tert-Butyldimethylsilyl
  • a solution that consists of 28 ml of diisobutyl aluminum hydride in 83 ml of toluene is added in drops at ⁇ 10° C. to a solution of 15.4 g of nitrile 4 in 280 ml of toluene.
  • the reaction solution is stirred at 0° C. until the reaction is completed, mixed in succession with 460 ml of toluene, 92 ml of saturated sodium bicarbonate solution, and 9 ml of 2-propanol, and it is stirred for several hours at room temperature. Then, it is filtered on Celite, and the filtrate is concentrated by evaporation.
  • a suspension that consists of sodium hydride (80%, 15 equivalents) in dimethyl sulfoxide (0.5 ml/mmol) is heated for 1 hour to 70° C. Then, a solution of the corresponding alkyltriphenylphosphonium bromide (15 equivalents) in dimethyl sulfoxide (2 ml/mmol) is added in drops at room temperature. The reaction solution is colored yellowish-green and is stirred for another hour at room temperature.
  • a solution that consists of 1 ml of diisobutylaluminium hydride in 1 ml of toluene is added in drops at ⁇ 10° C. to a solution of 1.16 g of cyanide 11a in 23 ml of toluene.
  • the reaction solution is stirred at ⁇ 10° C. until the reaction is completed, mixed in succession with 17 ml of toluene, 6 ml of saturated sodium bicarbonate solution and 0.7 ml of 2-propanol and stirred for several hours at room temperature. Then, it is filtered on Celite, and the filtrate is concentrated by evaporation.
  • a solution that consists of 0.4 ml of diisobutyl aluminum hydride in 0.4 ml of toluene is added in drops at ⁇ 10° C. to a solution of 480 mg of cyanide 11b in 10 ml of toluene.
  • the reaction solution is stirred at ⁇ 10° C. until the reaction is completed, mixed in succession with 8 ml of toluene, 3 ml of saturated sodium bicarbonate solution and 0.3 ml of 2-propanol, and stirred for several hours at room temperature. Then, it is filtered on Celite, and the filtrate is concentrated by evaporation.
  • a catalytic amount of tetrabutylammonium fluoride-trihydrate is added to a solution of the corresponding carbonyl compound (1 equivalent) in tetrahydrofuran (2 ml/mmol), the solution is cooled to ⁇ 20° C., and the (perfluoroalkyl)-trimethylsilane (15 equivalents) is added in drops. The cold bath is removed, and the reaction solution is stirred at room temperature until the reaction is completed. For working-up, it is diluted with diethyl ether, the organic phase is washed with water and saturated sodium chloride solution, dried on magnesium sulfate and concentrated by evaporation.
  • a corresponding amount of boron trichloride (1.5 equivalents each of ether that is to be cleaved, one additional equivalent for each basic grouping) is added in drops to a solution, cooled to ⁇ 78° C., of the corresponding steroid and tetrabutylammonium iodide (1.5 equivalents each of the ether to be cleaved, one additional equivalent for each basic grouping) in dichloromethane (5 ml/mmol).
  • the reaction solution is slowly heated to 0° C. and stirred until the reaction is completed.
  • the batch is mixed with ice water and stirred for about 30 more minutes, then it is mixed with saturated sodium bicarbonate solution and extracted several times with dichloromethane.
  • the combined organic phases are dried with magnesium sulfate and concentrated by evaporation.
  • the residue that is obtained is purified by column chromatography and yields the corresponding estradiols.
  • phase separation is carried out between diethyl ether/water, and the aqueous phase is extracted several times with diethyl ether.
  • the combined organic phases are washed with water and saturated sodium chloride solution, dried on magnesium sulfate and concentrated by evaporation.
  • Column-chromatographic purification (cyclohexane/ethyl acetate) yields 62 mg of estratrienediol 15a as a colorless, viscous mass (GC-MS: m/z theor.: 452, pract.: 452) and a mixture of its diastereomers D1/D2.
  • nButyllithium (1.6 M in hexane, 1 equivalent) is added in drops at ⁇ 78° C. to a 1 M solution of pentafluoroethyl iodide (10 equivalents in reference to the carbonyl compound), and the solution is stirred for 1 hour at ⁇ 78° C. Then, at this temperature, a solution of the corresponding carbonyl compound in tetrahydrofuran (5 ml/mmol) is added in drops. The reaction solution is slowly heated and stirred until the conversion is completed. For working-up, the reaction solution is mixed with saturated ammonium chloride solution and extracted several times with diethyl ether.
  • the colorless foam that is obtained is dissolved in 1 ml of toluene, 0.15 ml of diisobutyl aluminum hydride is added in drops at 0° C., and the reaction solution is refluxed until conversion is completed. After cooling to 0° C., the reaction mixture is mixed in succession with 0.5 ml of ethanol, 0.5 ml of ethanol/water (1:1) and 0.5 ml of semi-concentrated hydrochloric acid and stirred for about 30 more minutes. The phase separation is carried out between diethyl ether/water. The organic phase is washed with saturated sodium chloride solution, dried on magnesium sulfate and concentrated by evaporation.
  • 58 mg of pyridinium chlorochromate and 58 mg of Celite are introduced into 0.5 ml of dichloromethane, a solution of 95 mg of alcohol 25a in 0.9 ml of dichloromethane is added in drops, and the reaction mixture is stirred at room temperature until the reaction is completed. Then, the reaction mixture is filtered on Celite and silica gel, and the filtrate is concentrated by evaporation. This yields 81 mg of ketone 27a as a light yellow, viscous mass (GC-MS: m/z theor.: 524, pract.: 524), which is used without further purification in the reaction below.
  • GC-MS m/z theor.: 524, pract.: 524
  • a corresponding amount of bromide is dissolved in N,N-dimethylformamide (5 ml/mmol), mixed with an excess of the corresponding amine and stirred at room temperature (optionally at 40° C.) until the reaction is completed.
  • the phase separation is carried out between ethyl acetate/water, and the aqueous phase is extracted several times with ethyl acetate.
  • the organic phase is washed with water and saturated sodium chloride solution, dried with magnesium sulfate and concentrated by evaporation.
  • the column-chromatographic purification is carried out on silica gel with an ethyl acetate/methanol mixture as an eluant, and yields the corresponding amines.
  • the organic phase is washed with water and saturated sodium chloride solution, dried on magnesium sulfate and concentrated by evaporation.
  • the column-chromatographic purification is carried out on silica gel with a cyclohexane/ethyl acetate mixture as an eluant and yields the corresponding carboxylic acids.

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Lobaccaro C et al: "Steroidal Affinity Labels of the Estrogen Receptor 3, Estradiol 11.beta.-n-Alkyl Derivatives Bearing a Terminal Electrophilic Group: Anti-Estrogenic and Cytotoxic Properties" Journal of Medicinal Chemistry, American Chemical Society, Washington US, bd. 40, Nr. 14, Jul. 4, 1997.

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